Method of making antibiotics containing the aminocyclitol subunit

ABSTRACT

The method of making an antibiotic containing an aminocyclitol subunit. A microorganism mutant, incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an antibiotic when cultivated in a nutrient medium having no added aminocyclitol, is grown in the presence of an added aminocyclitol until substantial antibiotic activity is imparted to a culture medium, and the antibiotic is separated from the medium. Streptomyces fradiae ATCC 21401 mutant grown in the presence of added streptamine gives hybrimycins A1 and A2, and when grown in the presence of 2-epistreptamine gives hybrimycins B1 and B2. Streptomyces rimosus forma paromomycinus ATCC 21485 mutant grown in the presence of added streptamine gives hybrimycins C1 and C2. The hybrimycins have antibiotic activity.

United States Patent 1151 3,669,838 Shier et al. 1 June 13, 1972 54] METHOD OF MAKING ANTIBIOTICS 3,100,176 8 1963 Ehrlich et a1 ..195/80 x CONTAINING THE AMINOCYCLITOL 3,386,889 6/1968 Moses et al. ....195/80 x SUBUNIT 3,433,71 l 3/1969 Calbrider et al. nl95/80 X [72] Inventors: Wayne Thomas Shier; Kenneth L.

Rinehart, Jr.; David Gottlieb, all of Urbana,ll1.

[73] Assignee: University of Illinois Foundation, Urbana,

Ill

[22] Filed: March 17, 1970 [21] Applr No.: 20,433

Related U.S. Application Data [63] Continuation-impart of Ser. No. 842,712, July 17,

[52] U.S. Cl. ..195/29, 195/49, 195/80 R, 195/96, 260/210, 260/210 AB, 424/181 [51] Int.Cl. ..Cl2d 9/00 [58] Field ofSearch ..l95/80,81,49, 29, 96

[56] References Cited UNITED STATES PATENTS 2,916,485 12/1959 Frohardt et a1 ..260/210 AB 3,018,220 1/1962 McGuire et al. ....,195/8O X 3,022,228 2/1962 Moses ..195/80 Primary Examiner-Joseph M. Golian Attorney-Merriam, Marshall, Shapiro & Klose [5 7] ABSTRACT The method of making an antibiotic containing an aminocyclitol subunit. A microorganism mutant, incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an antibiotic when cultivated in a nutrient medium having no added aminocyclitol, is grown in the presence of an added aminocyclitol until substantial antibiotic activity is imparted to a culture medium, and the antibiotic is separated from the medium.

Streptomyces fradiae ATCC 21401 mutant grown in the presence of added streptamine gives hybrimycins A1 and A2, and when grown in the presence of 2-epistreptamine gives hybrimycins B1 and B2.

Streptomyces rimosus forma paromomycinus ATCC 21485 mutant grown in the presence of added streptamine gives hybrimycins C1 and C2,

The hybrimycins have antibiotic activity.

12 Claims, No Drawings work carried out, in part, under a grant or award from the Department of Health, Education and Welfare.

This invention relates to antibiotics. More particularly, this invention is concerned with the production of novel antibiotics by growing mutants of microorganisms which, in unmutated form, produce other antibiotics.

By growing various microorganisms, usually by submerged fermentation, a group of antibiotics has been obtained previously which contain an aminocyclitol group or subunit as part of the molecular structure. Among the aminocyclitol antibiotics which have been so produced are hygromycin, spectinomycin and the further defined antibiotics in which the aminocyclitol subunit is deoxystreptamine, including the antibiotics neomycin, paromomycin, kanamycin, gentamicin and nebrarnycin. The specific microorganisms used to produce such aminocyclitol-containing antibiotics are, in part, as follows:

Microorganism Antibiotic Srreptomyces fradiae and Slreptomyces albogn'sealus neomycin Streplomyce: kanamycen'cus kanarnycin Srrepwmyces rimosus forma paromomycinus Micmmonosporn echinorpom paromomycin and Micromonospom purpurea gentamicin Srreptomyces tenebrarius nebramycin Srrepromyces hygmscopicus and Slrepmmyces noboriloensi: hygromycin Srreptomyce: :peclabilir and Slrepromyce: flavopersicu: spectinomycin incorporates only one such subunit into an antibiotic. If an aminocyclitol other than the one normally formed by the microorganism could be microbiologically incorporated into the metabolic products, new products having antibiotic activity could be expected.

it has been found according to the present invention that mutants of microorganisms known to produce antibiotics containing an aminocyclitol subunit can be formed which lack the capacity to biosynthesize the aminocyclitol subunit but have the capacity to utilize an added aminocyclitol molecule to form an antibiotic. When the added aminocyclitol molecule is different than the aminocyclitol subunit present in the antibiotic produced by the unmutated microorganism, a new an tibiotic is produced. However, when the added aminocyclitol molecule is the same as the aminocyclitol subunit present in the old antibiotic produced by the unmutated microorganism, the mutated microorganism will produce the old antibiotic. The invention thus provides the method of making an antibiotic containing an aminocyclitol subunit, comprising cultivating or growing a microorganism mutant in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol, said mutant being incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an antibiotic when cultivated in a nutrient medium having no added aminocyclitol, continuing growing the mutant until substantial antibiotic activity is imparted to the culture medium and separating the antibiotic from the medium.

Mutants of antibiotic-producing microorganisms blocked in biosynthetic pathways so as to be incapable of forming the aminocyclitol subunit can be formed by treating conidia with a mutagen over a period of time long enough to permit germination and one or two generations, followed by random killing of cells, leaving among the survivors mutant daughter cells freed of their wild-type counterparts. Treatment with Z-arninopurine, S-bromouracil or proflavin, each followed by ultraviolet irradiation to achieve a high kill, or nitrosoguanidine alone, gives approximately the same percent auxotrophs among the survivors. For convenience, a nitrosoguanidine treatment is generally used.

A representative method of obtaining a desired mutant is to treat a monoconidial suspension of microorganism spores grown on a vegetable juice agar medium with nitrosoguanidine (0.5 mg/ml) in a basal glutamate medium to kill nearly all the microorganisms, inoculate Petri plates containing vegetable juice agar with the survivors, incubate until sporulation occurs and replica plate to Petri plates containing nutrient agar with and without an added aminocyclitol normally biosynthesized by the unmutated microorganism. Following incubation, the plates are overlaid with agar seeded with Bacillus subtilis or some other test organism, and then in cubated. The plates are then screened for a colony showing a zone of inhibition on nutrient agar containing the added aminocyclitol but no zone of inhibition on nutrient agar containing no added aminocyclitol. In this way, a suitable mutant is located alter which a stock culture of that organism is isolated from the master plate. The mutant so isolated will grow on a defined medium at approximately the same rate as the unmutated microorganism.

A deoxystreptamine-negative mutant of S. fradiae was isolated as described and in neomycin production showed a linear dependence on added deoxystreptamine at low concentrations with up to 73 percent conversion. This mutant has been deposited in the American Type Culture Collection on an unrestricted basis and has been given number ATCC, 21401.

Two deoxysueptamine-negative mutants of Streptomyces rimosus forma paromomycinus were also isolated as described. These mutants have been deposited in the American Type Culture Collection on an unrestricted basis and have been given numbers ATCC 21484 and 21485.

A deoxystreptamine-negative mutant of Streptomyces kanamyceticus was also isolated as described. This mutant has been deposited in the American Type Culture Collection on an unrestricted basis and has been given number ATCC 21486.

The invention also provides a process of growing the mutants so produced and isolated, in a nutrient medium containing an added aminocyclitol to produce one or more novel antibiotics. Processes for the isolation and recovery of some of the antibiotics are also provided. The medium used can be the same as is normally used to grow the unmutated microorganism, in the absence of an added aminocyclitol, and will contain at least water, a soluble carbohydrate, a source of assimilable nitrogen and essential mineral salts. Since the aminocyclitols are bases, they are readily addedas the free base or in the form of an acid addition salt such as the hydrochloride, sulfate or phosphate salts. Submerged fermentation on shakers for hours at 28 C. is usually employed for maximum growth. However, variations in growth conditions may be desirable from one mutant to another so the person skilled in the art should make adjustments to achieve the best results.

After growth is temiinated, the broth is filtered and is treated with a suitable anion exchange resin, advisably on the ammonium cycle, to absorb the antibiotic. The resin is then washed with ammonium hydroxide to elute the antibiotic. The eluates are concentrated under reduced pressure and then subjected to paper chromatography to separate and purify the antibiotic.

By growing the S. fradie mutant ATCC 21401 in this way in the presence of added streptamine, a mixture of two new antibiotics hybrimycins Al and A2 is obtained, while with added 2-epistreptamine (but no streptamine), a mixture of two new antibiotics hydrimycins Bi and B2 is obtained. By growing the S. nmosus forma paromomycinus mutant ATCC 21485 in the described way in the presence of added streptamine, a mixture of two new antibiotics hybrimycins Cl and C2 is obtained. The relationship of these new anti-biotics to each other is shown by the following formula.

Hybrimycin Al has the chemical name: [1S,2R,3R,4S,S R,6R]- l ,5-diamino-2-( 2,6-diamino-2,-dideoxy-a-D-glucopyranosyl )oxy-3-[ 3-0-( 2,6-diamino-2,-dideoxy-B-L- idopyranosyl )-}3-D-ribofuranosyl ]oxy-4,6-dihydroxycyclohexane. Hybrimycin A2 has the chemical name: lS,2R,3R,4S,5R,6R]- l ,5-diamino-2-(2,6-diarnino-2,6- dideoxy-a-D-glucopyranosyl )oxy-3- 3-0-( 2,6-diamino-2 ,6- dideoxy-a-D-glucopyranosyl)-B-D-ribofuranosyl]oxy-4,6 dihydroxycyclohexane. Hybrimycin Bl has the chemical name: [lS,2R,3R,4S,5R,6S1- l ,5-diamino-2-( 2,6-diamino-2,6- dideoxy-a-D-glucopyranosyl]oxy-3-[3-0-(2,6-diamino-2,6- dideoxy-B-L-idopyranosyl)-B-D-ribofuranosyl]xy-4,6- dihydroxycyclohexane. Hybrimycin B2 has the chemical name: lS,2R,3R,4S,5R,6S]-1,5-diamino-2-(2,6-diamino-2,6- dideoxy-a-D-glucopyranosyl )oxy-3-[ 3-O-( 2,6-diamino2,6- dideoxy-a-D-glucopyranosyl)-B-D-ribofuranosyl]oxy-4,6- dihydroxycyclohexane.

structurally, hybrimycins A l A2, Bl and B2 resemble neomycin except that the deoxystreptamine moiety has been replaced by another aminocyclitol. Also, similar to the unmutated microorganism s production of neomycins B and C is the production by the mutant, in each biosynthetic replacement of deoxystreptamine, of two new antibiotics. Hybrimycin Cl has the chemical name: lS,2R,3R,4S,5R,6R]-l ,S-diamino-2-( 2-amino-2-deoxy-a-D- glucopyranosyl )oxy- 3[ 3-0-( 2 ,6-diamino-2,6-dideoxy-B-L- idopyranosyl)-B-D-ribofuranosyl1oxy-4,fi-dihydroxycyclohexnc. Hybrimycin C2 has the chemical name: lS,2R,3R,4S,5R,6R]-1,5-diamino-2-(Z-amino-Z-deoxy-a-D- glucopyranosyl )oxy-3-[ 3-0-( 2 ,6-diamino-2,6-dideoxy-a-D- glucopyranosyl)-B-D-ribofuranosyl]oxy-4,6-dihydroxycyclohexane.

Structurally, hybrimycins Cl and C2 resemble paromomycin except that the deoxystreptamine moiety has been replaced by another aminocyclitol. Also, similar to the unmutated organism's production of paromomycins I and I1 is the production by the mutant of two new antibiotics each bearing the replacement of the deoxystreptamine moiety.

The S. kanamyceticur mutant ATCC 21486 has also been grown and antibiotics produced which are different than kanomycin.

ADDITIONAL DATA ON HYBRIMYCINS A l A2,Bl and B2 Hexa-N-acetylhybrimycins are readily produced by reacting a hybrimycin with acetic anhydride plus 3M dipotassium hydrogen phosphate according to the method of Pan and Dutcher, Anal. Chem. 28, 836 (1956). The hexa-N-acetylhybrimycins Al and A2, and hexa-N-acetylhybrimycins BI and B2, are readily separated and purified by paper chromatography in the solvent system l-butanol:pyridine:water:6: 4:3.

Optical rotational and R, differences between the hexa-N acetylhybrimycins A1 and A2, and between hexa-N-acetylhybrimycins B1 and B2, correspond closely to the differences between hexa-N-acetylneomycins B and C. The differences in optical rotation and R, values between hexa-N-acetylhybrimycin Al and hexa-N-acetylneomycin B correspond to the differences between the latter and hexa-N-acetylhybrimycin B1, and a similar relationship exists between hexa-N-acetylhydrimycins A2 and B2 and hexaN-acetylneomycin C. The dififerences are shown by the data in Table I. These relationships support the indicated stereochemistry of the hybrimycins A & B.

TABLE 1 Properties of N-Acetyl Derivatives a R, values are for paper chromatography in the solvent system l-butanol:pyridine:water:6:4: 3.

Hexa-N-acetylhybrimycins Al and B1 are readily converted to octa-0-trimethylsilyl-hexa-N-acetylhybrimycins Al and B1 by reacting with N,0bis-(trimethylsilyl)-acetamide in anhydrous pyridine. The mass spectra of these derivatives show molecular ions at m/e 1458, the expected molecular weight, as well as fragments at m/e 389 and 665 corresponding to the non-arninocyclitol moieties of the antibiotics. The nuclear magnetic resonance spectra of hybrimycins A and B show no absorption by methylene protons (ABX multiple!) characteristic of the deoxystreptamine moiety of neomycin. This establishes that the hybrimycins A & B have the indicated structure.

The antibacterial activity of the hybrimycin A and hybrimycin B complexes or mixtures and of hybrimycins Al, A2, B1 and B2 against various test organisms is illustrated by the following data.

1. ANTIBACTERIAL ACTIVITY OF COMPLEXES OF HYBRIMYCINS A AND B:

These antibiotic complexes were dissolved in brain heart infusion broth and twofold decrements were made from 200 pig/ml. An 18-hour culture of each test organism was diluted 1:2,000 and one drop of the diluted suspension was added to 1 ml of broth with the antibiotic. The test system was estimated to contain 10 organisms per ml. All tubes were incubated at 37 C. for 24 hours. The minimum inhibitory concentration is the lowest concentration of antibiotic which prevented growth. The results are given in Table 11.

TABLE 11 Antibacterial Activity of I-iybrimycins A and B OSU, Ohio State University; UC, Upjohn Co.; ATCC, American Type Culture Collection. Minimum inhibitory concentration.

2. ANTIBACTERIAL ACTIVITY OF HYBRIMYCINS A1, A2, B1 and B2 The potency of hybrimycin A1, A2, B1 and B2 preparations was compared to that of neomycin by use of the standard agar diffusion plate assay procedure. The test organism used was Escherichia coli ATCC I 1775. Bio-assay agar (medium A) having the composition:

Beef extract 1.5 g Yeast 3.0 g Peptone =6.0 g Agar 15.0 g Water 1000 ml was autoclaved for 20 minutes at 120 C., cooled to 46 C., and inoculated with 5 ml of a suspension of late log phase cells of the test organism in the following medium (medium B), per 100 ml of the bio-assay agar (medium A);

Grams Beef extract 0.4 Cerelose 1.0 Peptone 0.4 Yeast extract 0.1 Sodium chloride 0.25

A volume of 8 ml of the inoculated medium was poured into each of a series of Petri dishes. Filter paper discs 12.7 mm in diameter) were dipped in aqueous solutionsof the material to be assayed and the disc was placed on the surface of the agar. The plates were stored at zero to 4 C. for 2 hours and then incubated 12 to 16 hours at 28 C. Zone sizes at various concentrations from 5 to 100 ag/ml were plotted against the log of the concentration of each antibiotic to afford standard curves from which the concentration of hybrimycins in various preparations could be determined by comparison. The following Table 111 gives the data derived from the semllog plots.

TABLE III Antibionc Concentration of Antibiotic At Same Zone Size-Produced by 5 pig/ml Neomycin B l-Iybrimycin A1 19.7 pg/ml A2 23.8 lg/ml 51 47.5 m! B2 66.8 palm] ADDITIONAL DATA ON I-IYBRIMYCINS C1 AND C2 Penta-N-acetylbybrimycins C1 and'C2 are prepared by the method described for the preparation of hexa-N-acetylhybrimycins Al A2, B1 and B2.

Optical rotations and Rf values of penta-N-acetylhybrimycins Cl and C2 correspond to those of penta-N-acetylparomomycins I and 11 except that the specific rotations of hybrimycin Cl and C2 are less dextrorotatory as shown in the following Table IV.

R, values are for paper chromatography in the solvent system l-butanol:pyridine:water: 6:4:3.

Since hexa-N-acetyl-hybrimycins Al and A2 are less dextrorotatory than hexa-N-acetylneomycins B and C, respectively, due to the presence of an additional hydroxyl group in the 6R configuration, the additional hydroxyl group in hybrimycins Cl and C2 must also be in the 6R configuration.

Penta-N-acetyl hybrimycins Cl and C2 are readily converted to nona-O-trimethylsilyl-penta-Nacetylhybrimycins Cl and C2 by reaction with N,O-bis-(trimethylsilyl)-acetamide in anhydrous pyridine-N-,N-dimethylformamide. The mass spectra of these derivatives show molecular ions at m/e 1489, the expected molecular weight, as well as fragments at m/e'389, 420 and 665, corresponding to non-aminocyelitol moieties of the antibiotics. The nuclear magnetic resonance spectrum of hybrimycin C shows no absorption by methylene protons (ABX multiplet) characteristic of the deoxystreptamine moiety of paromomycin. This establishes that hybrimycins C1 and C2 have the indicated structures.

The antibacterial activity of hybrimycins Cl and C2 against various test organisms is illustrated by the following data.

1. Hybrimycins Cl and C2 were dissolved in brain heart infusion broth and twofold decrements. were made from 200 ag/ml. An 18-hour culture of each test organism was diluted 122,000 and one drop of the diluted suspension was added to 1 ml. of broth with the antibiotic. The test system was estimated to contain 10" organisms per ml. All tubes were incubated at 37 C for 24 hours. The results are given in Table V.

TABLE V Antibacterial Activity of Hybrimycins Cl and C2 Test Oganism" MIC (pg/ml) Paromo- Hybri- Hybromycin mycin C l mycin C2 Escherichia coli ATCC 26 17.8 25.0 50.0

ATCC, American Type Culture Collection; UC, Upjohn Co. Minimum inhibitory concentration.

2. The comparative antibacterial activity of paromomycin and hybrimycins Cl and C2 against a strain of Bacillus subtilis was determined. The antibiotic preparations were dissolved in the following medium (Medium L) and twofold decrements were made from 200 pig/ml.

Peptone Beef extract Yeast extract Glucose Sodium chloride Dipotassium monohydrogen phosphate 3.

Potassium dihydrogen phosphate Water 1.32 g 1000 ml A growing culture of the test organism was diluted 1:2,000 and one drop of the diluted suspension was added to l m1. of broth with the antibiotic. All tubes were incubated at 37 C for 24 hrs. The results are reported in Table VI. The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotic which prevented growth.

TABLE VI Antibacterial Activity of Hybrimycins Cl and C2 MIC (I- -SJmI.)

Test Organism Paromomycin Hybrimycin Cl Hybrimycin C2 Bacillus subrilis 6.25 25 .0 50.0

EXAMPLE 1 Preparation of S. fradiae Mutant ATCC 21401 A mutant of the organism Strepromyces fradiae was prepared by treatment of S. fradiae 3535 with N-methyl-N'- nitro-N-nitrosoguanidine. A slant containing 5 ml of the following medium (medium C) Mixed vegetable juice i 20 ml Calcium carbonate 0.3 g. Agar 2.0 g.

Distilled water to 100 ml was inoculated from soil stock of S. fradiae 35 35. After incubation for 7 days at 28 C., spores from a slant were suspended in water and transferred to 100 ml of the following medium (medium D) in a 500 ml Erlenmeyer flask:

Grams Glucose 5 L-Glutamic acid 10 Dipotassium hydrogen phosphate 1.5 Magnesium sulfate heptahydrate 0.025 Calcium chloride 0.025 Ferrous sulfate heptahydrate 0.015

Zinc sulfate heptahydrate Distilled water to 1000 ml Prior to the inoculation, medium D had been adjusted to pH 7.0 with potassium hydroxide and autoclaved 20 minutes at 120 C. The glucose had been autoclaved separately as a 50 percent stock solution. To a flask containing the above inoculated medium (D), was added 50 mg of N-methyl-N'-nitro-N- nitrosoguanidine, and the vessel was incubated 12 hours at 28 C. with reciprocal shaking. Petri plates containing 10 ml of the V-8 agar (medium C) were inoculated with a diluted suspension of the organism in the flask and incubated at 28 C. until sporulation occurred. Spores were transferred by the replica plating technique to Petri plates containing 10 ml of the following medium (medium E):

Grams Peptone 0.5 Beef extract 0.4 Sodium chloride 0.5 Agar I .5

Tap water to ml The pH was adjusted to 7.2 -7.4 and the medium (E) was autoclaved for 20 minutes at C.

Spores from the same V-8 agar plate were also transferred by the same technique to Petri plates containing the same medium plus 200 pg/ml of 2-deoxystreptamine dihydrobromide. The Petri plates were incubated 44 hours at 28 C. at which time 8 ml of another medium (medium F) was added to the plates. Medium F was Bacto Streptomycin assay agar (medium A) sterilized at 120 C. for 20 minutes, cooled to 46 C. and mixed with 5 ml of late log phase cells of Bacillus subtilis per 100 ml of the assay agar.

The plates were stored at 0 C. for l hour and incubated at 28 C. for 14 hours. The plates were examined for a colony that showed a zone of inhibition on the medium containing 2- deoxystreptamine dihydrobromide but no zone of inhibition on the medium lacking 2-deoxystreptamine dihydrobromide. The mutant ATCC 21401 was isolated from the V-8 agar (medium C) plate.

The so-isolated deoxystreptamine-negative mutant grows in a defined medium at approximately the same rate as the unmutated S. fradiae. This mutant has been deposited in the American Type Culture Collection on an unrestricted basis and has been given number ATCC 21401. Antibiotic production showed a linear dependence on added deoxystreptamine at low concentrations with up to 73 percent conversion of added deoxystreptamine dihydrobrornide as determined by assay against B. subtilus.

ANTIBIOTIC PRODUCTION WITH THE S. F RADIAE MUTANT ATCC 2 I401 By growing the mutant ATCC 21401 in a nutrient medium containing the aminocyclitol streptamine, a mixture of two new antibiotics, hybrimycins A1 and A2, are obtained. When the medium contains the aminocyclitol Z-epistreptamine, a mixture of two other new antibiotics, hybrimycins B1 and B2, are obtained. There follow examples to illustrate the production, recovery, concentration, purification, and structural proof of these four hybrimycin antibiotics.

EXAMPLE 2 Production of l-Iybrimycins Al and A2 and Recovery To produce hybrimycins A, test tube slants were prepared, each containing 5 ml of medium C. The slants were autoclaved at 120 C. for 20 minutes and allowed to set at a 30 angle. A slant was inoculated with spores of the deoxystreptamine-negative mutant S. fradiae ATCC 21401 from a soil stock culture and incubated 7 days at 28 C. An aqueous suspension (5 m1) of spores from this slant was used to inoculate a 500 ml Erlenmeyer flask containing 100 ml of the following seed medium (medium G):

Grams Glucose l Hydrolyzed casein (N-Z- Amine A), (SHEFFIELD Farms, inc.) 1 Diammonium phosphate,

anhydrous 0.5 Magnesium sulfate heptahydrate 0.05 Cupric sulfate, pentahydrate 0.005 Ferrous sulfate, heptahydrate 0.005 Calcium carbonate 1 Tap water to 100 ml Grams Glucose l Soybean meal 2.5 Yeast extract 0.5 Sodium chloride 0.5 Calcium carbonate 0.2 streptamine hydrosulfate 0.025

Distilled water to 100 ml Prior to inoculation, the medium was adjusted to pH 7.0 7.1 with potassium hydroxide solution and autoclaved 20 minutes at 120 C.

The vessels containing the production medium were incubated for 120 hours at 28 C. with reciprocal shaking. .After the indicated period of time, the beer was filtered through milk filters and 2 liters of the cloudy filtrate was twice passed slowly down a column of 50 ml of a weakly acidic, carboxylic (polymethacrylic) type cation exchange resin of medium porosity (Amberlite [RC-50) in the ammonium form. The spent beer was discarded, the column was washed with 4 liters of deionized water and eluted with 200 ml of 2 N ammonium hydroxide solution. The eluate was evaporated under reduced pressure (0.2 mm), the residue was dissolved in a small portion of water, applied to chromatographic paper (Whatman 3 MM paper) and chromatographed with absolute methanokconcentrated ammonium hydroxide:4:1. The location of the antibiotic was visualized on strips cut from the chromatogram by spraying in succession, followed by air drying, with aqueous sodium hypochlorite 1 percent), twice with 95 percent ethanol and finally with a saturated solution of benzidine in 2 percent aqueous acetic acid containing a crystal of potassium iodide. The segments of paper corresponding to the major blue spot were cut out and eluted with deionized water. The eluate was lyophilized to yield 196 mg of a white, amorphous powder with antibacterial activity. The white powder was a mixture of hybrimycin A1 and hybrimycin A2.

Hybrimycin Al was separated from hybrimycin A2 by chromatography on paper as described in the previous paragraph except that the leading one-third of the paper segment corresponding to the blue spot was eluted separately to yield hybrimycin A1; similarly the trailing one-third yielded hybrimycin A2. The remainder of the material was eluted and rechromatographed.

Hexa-N-acetylhybrimycins A1 and A2 were prepared by reacting a mixture of hybrimycins A1 and A2 with acetic anhydride in the presence of a 3 molar dipotassium hydrogen phosphate solution according to the method of Pan and Dutcher, Anal. Chem., 28, 836 (1956). Hexa-N-acetylhybrimycins Al and A2 were separated andpurified by paper chromatography using the solvent system l-butanol:pyridine:water:6:4:3.

EXAMPLE 3 Production of Hybrimycins B1 and B2 and Recovery A mixture of hybrimycins Bl and B2 was produced using the same mutant ATCC 21401 and in the same manner as hybrimycins A1 and A2 in Example 2 except that the production medium (medium H) contained Z-epistreptamine dihydrochloride (0.025 gram per ml of medium) and contained no streptamine hydrosulfate. Two liters of incubated production medium so obtained yielded 56 mg of a white, amorphous powder with antibacterial activity. The powder was a mixture of hybrimycin B1 and hybrimycin B2. Hybrimycin Bl was separated from hybrimycin B2 in the same manner as hybrimycin Al was separated from hybrimycin A2 in Example 2.

Hexa-N-acetylhybrimycins B1 and B2 were prepared by reacting a mixture of hybrimycins B1 and B2 with acetic anhydride in the presence of a 3 molar dipotassium hydrogen phosphate solution according to the method of Pan and Dutcher, Anal. Chem., 28, 836 (1956). Hexa-N-acetylhybri mycins B1 and B2 were separated and purified by paper chromatography using the solvent system l-butanol:pyridine:water:6:4:3.

EXAMPLE 4 Preparation of S. rimosus forma paromomycinus ATCC 21484 and 21485 Two mutants of the organism Streptomyces rimosus forma I paromomycinus were prepared by treatment of S. rimosus forma paromomycinus ATCC 14827 with N-methyl-N-nitro- N-nitrosoquanidine. A slant containing 5 ml. of medium C was inoculated from a soil stock of S. rimosus forma paramomycinus ATCC 14827. After incubation for at least 7 days at 26 C, spores from a slant were suspended in water and transferred to 100 ml. of sterile medium L supplemented with 3 mg. of N-methyl-N-nitro-N-nitrosoguanidine, and the vessel was incubated for 12 hours at 26 C. with reciprocal shaking. Aliquots of this broth were treated in the manner described for the isolation of the deoxystreptamine-negative mutant of S. fradiae (ATCC 21401). Two deoxystreptamine negative mutants of S. n'mosus forma paromomycinus were isolated from the medium C plate, and deposited in the American Type Culture Collection on an unrestricted basis and given numbers ATCC 21484 and 21485.

ANTIBIOTIC PRODUCTION WITH THE S. RIMOS US FORMA PAROMOM YCIN US MUTANT ATCC 21485 EXAMPLE 5 Production of Hybrimycins Cl and C2 and Recovery.

To produce hybrimycins Cl and C2, test tube slants were prepared, each containing 5 ml of medium C (Example 1). The slants were autoclaved at C. for 20 minutes and allowed to set at a 30 angle. A slant was inoculated with spores of the deoxystreptamine-negative mutant of S. rimasus forma myer flask containing 100 ml. of the following seed medium (medium .1 )2

Grams Cerelose 1.0 Soybean meal 1.0 Sodium chloride 0.5 Calcium carbonate 0.5 Ammonium chloride 0.167 Casein 0.25

Tap water to 100 ml Prior to inoculation, the medium was adjusted to pH 7.5 with sodium hydroxide solution and flasks of the medium were autoclaved at 1 20 C. for 20 minutes.

The flask containing medium .1 was incubated at 26 C. for 64 hrs. with reciprocal shaking, and 7.5 ml aliquots were used to inoculate 100 ml. portions of the production medium (medium K) in 500 ml shake flasks. Medium K contains the components of medium J plus 40 mg of streptamine hydrosulfate per 100 ml. The production flasks were incubated at 26 C for 5 days with reciprocal shaking. The contents of the flasks were pooled and the beer was filtered through cotton cloth. The filtrate was treated in the same manner as the production broths of the other hybrimycins. From 2 liters of filtrate, 82 mg. of a white amorphous powder with antibacterial activity was obtained. The white powder was a mixture of hybrimycins C1 and C2.

l-lybrimycin C1 was separated from C2 as described for the separation of hybrimycins A1 and A2, and B1 and B2.

Penta-N-acetylhybrimycins Cl and C2 were prepared, separated and purified by the procedure described for hexa-N- acetylhybrimycins Al and A2.

The mutants ATCC 21484 and 21485 appear to be very similar. By growing the mutant ATCC 21484 in a nutrient medium containing the aminocyclitol streptamine, an antibiotic substance was also produced.

EXAMPLE 6 Preparation of S. kanamyceticus ATCC 21486 A mutant of the organism Streptomyces kanamyceticus was prepared by treatment of S. kanamyceticus ATCC 12853 with N-methyl-N'-nitro-N nitrosoguanidine. Slants containing 5 ml. of medium C were inoculated from a soil stock of S. kanamyceticus ATCC 1285 3. After incubation for at least 5 days at 26 C, spores from a slant were suspended in water and transferred to 100 ml. of sterile medium L supplemented with 2 mg. of N-methyl-N-nitro-N-nitrosoguanidine, and the vessel was incubated for 6 hours at 26 C with reciprocal shaking. After incubation, 1 ml. aliquots were diluted l-fold in sterile water, and 1 ml. aliquots of the diluted suspension were transferred to slants containing ml. of medium C and incubated at 26 C for at least 5 days. The growth from one of these slants was transferred to 100 ml. of sterile water and blended in a blender for 8 min. One ml. aliquots of this suspension were subjected to serial dilution through three tubes of 9 ml. of sterile water. Aliquots of the final dilution were treated in the manner described for the isolation of the deoxystreptamine-negative mutant of S. fradiae (ATCC 21401). A deoxystreptaminenegative mutant of S. kanamycericus was isolated from a medium C plate and deposited in the American Type Culture Collection on an unrestricted basis and given number ATCC 21486.

12. EXAMPLE 7 Fermentation of S. kanamyceticw Mutant ATCC 21486.

The mutant of Streptomyces kanamycetieus ATCC 21486 was grown in streak cultures in 40 Petri dishes containing 10 ml. of medium E supplemented with 25 mg of Z-epistreptamine dihydrochloride per ml of medium E for 72 hr. at 26 C. The contents of the Petri plates were pooled, frozen, thawed, filtered, and the filtrate passed down a column of 25 ml of a weakly acidic carboxylic (polymethacrylic) type cation exchange resin of medium porosity (Amber-lite [RC-50) in the ammonium form. The 50) filtrate was discarded, the column washed with 1 liter of deionized water, and eluted with 100 ml of 2N ammonium hydroxide solution. The eluate was evaporated under reduced pressure (0.2 mm), the residue dissolved in 1.0 ml of water, and 100 pl applied to chromatographic paper (Whatrnan No. l). A sample of 20 pg of authentic kanarnycin (Kantrex, Bristol) was applied to the same paper, and the materials chromatographed with absolute methanol: concentrated ammonium hydroxide solution: 4:1. The location of the antibiotics was visualized by placing the chromatograph on the surface of ml of medium A that had been autoclaved for 20 min.,' cooled to 48 C and mixed with 5 ml of a suspension of spores of Bacillus subtilir in medium B, then allowed to set in an 8 by 14 inch dish. After 4 min. the paper was removed, and the medium incubated at 26 C for 20 hr. Zones of inhibition of growth of B. subtilis indicate the location of antibiotic on the original chromatogram. The antibiotic from the mutant of S. kanamycetieus has a lower R value (0.18) than that of kanamycin (R, 0.54), indicating the new antibiotic is similar in structure, but is more polar, consistent with the presence of an additional hydroxyl group.

The antibiotics of this invention are useful antimicrobial agents. Amongst the uses, is their use as topical agents to render surfaces sterile or to substantially reduce the presence of bacteria thereon.

What is claimed is:

1. The method of making an antibiotic containing an aminocyclitol subunit which comprises:

cultivating a microorganism'mutant in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol;

said mutant being incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an an tibiotic when cultivated in a nutrient medium having no added aminocyclitol;

continuing growing the mutant until substantial antibiotic activity is imparted to the culture medium; and

separating the antibiotic from the medium.

2. The method of claim 1 in which the mutant is incapable of biosynthesizing deoxystreptamine, and deoxystreptamine and the added aminocyclitol have dissimilar groups.

3. The method of making an aminocyclitol-containing antibiotic which comprises:

cultivating a mutant of (a) Streptomycesfradiae, (b) Streptomyces albagriseolus, (c) Strepromycer kanamyceticus, (d) Streptomyces rimosus forma paromomycinus, (e) Micromonospora echinospora, (f) Micromonospora purpurea, (g) Streptomyces renebrarius, (h) Streptomyces hygroscopicus, (i) Streptomyces noboritoensis, (j) Streptomyces sp ectabilis, and (k) Streptomyces flavopersicus, in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol;

said mutant being incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an antibiotic according to the correlation;

Unrnutated Microorganism Antibiotic (a) and (b) neomycin kanamycin paromomycin (e) and (f) gentamicin (g) nebramycin (h) and (i) hygnomycin (j) and spectinomycin when cultivated in a nutrient medium having no added aminocyclitol;

continuing growing the mutant until substantial antibiotic activity is imparted to the culture medium; and, separating the antibiotic from the medium. 4. The method of making an antibiotic containing an aminocyclitol subunit which comprises:

cultivating the microorganism Streptamyces fi'adiae ATCC 21401 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol; continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium; and separating the antibiotic from the medium. 5. The method of claim 4 in which the added aminocyclitol is streptamine.

6. The method of claim 4 in which the added aminocyclitol is 2-epistreptamine.

7'. The method of claim 5 in which the antibiotic produced is a mixture of hybrimycin A1 and hybrimycin A2.

14 8. The method of claim 6 in which the antibiotic produced is a mixture of hybrimycin B1 and hybrimycin B2.

9. The method of making an antibiotic containing an aminocyclitol subunit which comprises:

5 cultivating the microorganisms Streptomyces rimosus forma paromomycinus ATCC 21484 or 21485 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol;

continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium; and separating the antibiotic from the medium. 10. The method of claim 9 in which the added aminocyclitol is streptamine.

11. The method of making an antibiotic containing an aminocyclitol subunit which comprises:

cultivating the microorganism Strcptomyces kanamyceticus ATCC 21486 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol; and

continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium. 12. The method of claim 1 in which the added aminocyclitol is 2-epistreptamine. I I fi l mg UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,669,838 Dated vMarch 17 1970 Wayne Thomas Shier, Inventor(s) Kenneth L. Ri'nehart Jr. and David Gottliejb It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 3, line 25, R2 should be"'R column 5, line 20, 5.00 should be "50.00"; column 5, line 50, =6.0

8 should be "6.0g"; column 12, line 13, $0} should be "spent".

Signed and sealed this 10th day of October 1972.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Commissioner of Patents Attesting Officer 

2. The method of claim 1 in which the mutant is incapable of biosynthesizing deoxystreptamine, and deoxystreptamine and the added aminocyclitol have dissimilar groups.
 3. The method of making an aminocyclitol-containing antibiotic which comprises: cultivating a mutant of (a) Streptomyces fradiae, (b) Streptomyces albogriseolus, (c) Streptomyces kanamyceticus, (d) Streptomyces rimosus forma paromomycinus, (e) Micromonospora echinospora, (f) Micromonospora purpurea, (g) Streptomyces tenebrarius, (h) Streptomyces hygroscopicus, (i) Streptomyces noboritoensis, (j) Streptomyces spectabilis, and (k) Streptomyces flavopersicus, in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol; said mutant being incapable of biosynthesizing the aminocyclitol molecule but which molecule the unmutated microorganism biosynthesizes in the formation of an antibiotic according to the correlation; Unmutated Microorganism Antibiotic (a) and (b) neomycin (c) kanamycin (d) paromomycin (e) and (f) gentamicin (g) nebramycin (h) and (i) hygromycin (j) and (k) spectinomycin when cultivated in a nutrient medium having no added aminocyclitol; continuing growing the mutant until substantial antibiotic activity is imparted to the culture medium; and, separating the antibiotic from the medium.
 4. The method of making an antibiotic containing an aminocyclitol subunit which comprises: cultivating the microorganism Streptomyces fradiae ATCC 21401 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogEn, essential mineral salts and an added aminocyclitol; continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium; and separating the antibiotic from the medium.
 5. The method of claim 4 in which the added aminocyclitol is streptamine.
 6. The method of claim 4 in which the added aminocyclitol is 2-epistreptamine.
 7. The method of claim 5 in which the antibiotic produced is a mixture of hybrimycin A1 and hybrimycin A2.
 8. The method of claim 6 in which the antibiotic produced is a mixture of hybrimycin B1 and hybrimycin B2.
 9. The method of making an antibiotic containing an aminocyclitol subunit which comprises: cultivating the microorganisms Streptomyces rimosus forma paromomycinus ATCC 21484 or 21485 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol; continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium; and separating the antibiotic from the medium.
 10. The method of claim 9 in which the added aminocyclitol is streptamine.
 11. The method of making an antibiotic containing an aminocyclitol subunit which comprises: cultivating the microorganism Streptomyces kanamyceticus ATCC 21486 in an aqueous medium containing a soluble carbohydrate, a source of assimilable nitrogen, essential mineral salts and an added aminocyclitol; and continuing growing the microorganism until substantial antibiotic activity is imparted to the culture medium.
 12. The method of claim 1 in which the added aminocyclitol is 2-epistreptamine. 